Phase Ⅰ Study Of Low Dose Radiation Combined With Sbrt And Anti-PD-1 In Metastatic NSCLC And Effect Of Radiation Immune Homing

Background:Radiotherapy is a wildly treatment for local or oligometastatic tumors.Tumor cells exposure after irradiation release the "eat me" signal,promoting dendritic cells cell maturation and antigen presentation,which could activate the effector T cells.Irradiation site also release chemokines and cytokines to recruit T cells trafficking into the tumor.At the same time radiation could induce immune suppression with increasing the Programmed death receptor-1(PD-1)/Programmed death ligend-1(PDL1)expression and transforming growth factor-β(TGF-β).PD-1 inhibitor relieves and delays T cell apoptosis,thus delaying distant tumor growth.Stereotactic radiotherapy(SBRT)could decrease myeloid-derived suppressor cells(MDSC)and Treg cell recruitment and protect lymph node,at present,PD-1 inhibitors combined with SBRT therapy is the most anticipated immunotherapy,however preclinical and clinical evidence shows that single-site SBRT combined with PD-1 inhibitors is not ideal for the control of distant tumors.Based on our previous basic research,it was found that the triple therapy mode of SBRT combined with PD-1 inhibitor combined with low dose radiotherapy for distant lesions could increase the control of radiotherapy combined with immunity on the growth of tumors in situ and distal sites.Retrospective studies also showed that patients with advanced non-small cell lung cancer(NSCLC)still responded to the triple therapy mode after multi-line therapy.Therefore,we conducted this phase I clinical trial to explore the safety of low-dose radiotherapy combined with SBRT and PD-1 inhibitors in patients with advanced PD-L1-positive NSCLC,we also preliminarily evaluate the efficacy of triple therapy and dynamically monitor the changes of peripheral blood circulating immunity after triple therapy.The anti-tumor immune effect depends on the activation and recruitment of CD8 +T cells into the tumor to produce cytotoxic molecules(such as perforin and granzyme)and cytokines(such as IFN-γ and TNF-α).Previous studies have shown that while radiotherapy promotes tumor cell antigen exposure,it also releases a large number of chemokines to attract more T cells to recruit into the tumor and play a cytotoxic role.Interferon-induced chemokine CXCL9,CXCL10 and CXCL11 bind to activated T cell surface chemokine receptor CXCR3,which is an important chemokine factor in inducing T cell migration and effector function.The previous study of our group observed the increase of T cells in the tumor after low-dose radiotherapy,so this study further explored whether low-dose radiotherapy can recruit T cells into the tumor by up-regulating the expression of chemokine.Objective:1.Explore the safety of low-dose radiotherapy combined with SBRT and PD-1inhibitors in patients with advanced PD-L1-positive NSCLC in first-line,and to evaluate the effectiveness of triple therapy.2.Monitoring the dynamic changes of peripheral blood circulating immunity and tumor burden after triple therapy.3.Explore the possible mechanism of increased intratumoral T cell recruitment after low dose radiotherapy.Providing a theoretical basis for the anti-tumor effect of lowdose radiotherapy combined with SBRT and PD-1 inhibitors.Materials and methods:1.This study is a phase I clinical trial of dose exploration and dose expansion.The purpose of this study is to observe and evaluate the tolerance of low-dose radiotherapy combined with SBRT and PD-1 inhibitors,and to determine the recommended clinical dose of low-dose radiotherapy.At the same time investigating the safety and efficacy of triple therapy as first-line treatment of PD-L1-positive NSCLC patients in stage IV.Patients with tumor sites(≥2 lesions)available for SBRT and low dose radiotherapy were inclusion.In Ia phase,the safe dose of low dose radiotherapy was determined by3+3 dose climbing mode.SBRT and PD-1 inhibitors were fixed dose.Small lesion(diameter ≤3cm)was treated with SBRT.On the same day,large lesion(diameter >small tumor diameter)was treated with low dose radiotherapy.Within one week after radiotherapy,PD-1 inhibitors were administration every 21 days within one week after radiotherapy completion for no more than 24 months,or until the disease progressed and the trial was terminated,or the toxicity was not tolerated.According to the dose limit toxicity(DLT)cases of combination therapy,the tolerance of low dose radiotherapy dose climbing(2Gy/1f,4Gy/2f,10Gy/5f)combined with SBRT and PD-1 inhibitor treatment was observed.The observation time of DLT was 28 days(±3days)after the beginning of combined therapy.In dose escalation stage,the group could expand 3 patients in parallel to evaluate the treatment response if more than 1patients evaluate partial response.In Ib stage,the expansion dose of low dose radiotherapy was determined according Ia stage and further evaluating the safety and effectiveness of triple therapy.The objective response rate(ORR)and progressionfree survival(PFS)was recorded.2.Peripheral blood was also collected to monitor the dynamic changes of circulating tumor immunity,including peripheral blood neutrophils/lymphocytes(N/L)ratio,eosinophils percentage and lactate dehydrogenase(LDH)at baseline,after radiotherapy,6 weeks and 12 weeks after PD-1inhibitor.The classification proportion of T cells,Treg cells and PD-1+T cells in peripheral blood were monitored by flow cytometry.The dynamic monitoring of circulating tumor cells(CTC)and circulating tumor DNA(ct DNA)were used to understand the changes of peripheral circulating tumor burden,and T cell receptor(TCR)sequencing was used to monitor the diversity changes of peripheral blood T cell receptor.3.Single CT26 and LLC tumor model with low dose therapy was established to explore the mechanism of low dose radiotherapy on T cell recruitment.Transcriptome sequencing and fluorescence quantitative PCR were used to verify the upregulation chemokines in the tumor after low dose radiotherapy.The effect of chemokine receptor inhibitors(αCXCR3)on T cell recruitment after low-dose radiotherapy was detected with flow cytometry,immunofluorescence and western blot analysis.At the same time,the tumor volume growth curve were also recorded.Result:1.This trail was approved with clinical ethics committee of West China hospital.Phase Ia included 18 patients,phase Ib included 3 patients.The median follow-up time was 12.5 months(range: 1.1-21.3 months)on cutoff day February 8,2021.All patients completed radiotherapy and at least one cycle of PD-1 inhibitor.No treatment-related DLT occurred during dose climbing stage.No serious adverse events occurred.Treatment-related adverse events(AE)were observed in 90.5% patients.Grade 1 AE were observed in 85.7%patients,grade 2 AE were observed in 38.1% patients.Grade3 AE were observed in 23.8% patients.The main grade 3 AE were pneumonia,immune-related rash and immune-related triglyceride increase.No patients experienced grade 4 AE.PR was observed in 12(57.1%)patients,SD was observed in 6(26.8%)patients,and PD was observed in 3(14.3%)patients.No CR patients.The overall ORR was 57.1 %,and the disease control rate was 85.7 %.The overall measurable lesions were reduced by an average of 44.6 %(in 17 patients),and the lowdose radiotherapy lesions were reduced by an average of 50.3 %(in 15 patients).A total of 11 lesions were observed in 5 patients.The median PFS was 9 months(range:1.1-18.2,95 % CI: 5.7-12.4).PFS with 3 months was 81%,PFS with 6 months was62.7%,PFS with 12 months was 36.7%.2.After triple therapy,LDH decreased significantly(baseline vs 12 weeks:209.3IU/L vs 172IU/L,p<0.05),PD-1+T cell percentage decreased significantly(baseline vs 12weeks: 6.5% vs 0.42%,p<0.001),Treg cell percentage decreased significantly(baseline vs post radiation: 0.98% vs 0.47%,p<0.05),and mean VAF of ct DNA decreased significantly(baseline vs 6 weeks: 0.029 vs 0.011,p<0.05).while CD8 + T cell increased significantly(baseline vs 12 weeks: 29.3% vs 36.3%,p<0.05).Survival analysis showed that low LDH and N/L ratio had longer PFS(high vs low:1.7 months vs 11.8 months,p<0.01,HR: 0.2091,95%CI: 0.004-0.417),high eosinophil percentage patient showed longer PFS(high vs low:not reach vs 5.7 month,p<0.05,HR: 4.387,95%CI: 1.382-15.81),low PD-1+T cell expression showed longer PFS(high vs low:5.7 months vs not reach,p<0.01,HR: 0.1642,95%CI:0.059-0.644),low TMB showed longer PFS(high vs low:5.74 months vs not reach,p<0.05,HR: 0.288,95%CI: 0.086-0.938)and low ct DNA predicted prolonged PFS((high vs low:5.2 months vs not reach,p<0.05,HR: 0.2732,95%CI: 0.08522-0.9334).High TCR clone counts showed longer PFS(high vs low:not reach vs 4.4months,p < 0.05,HR: 3.465,95%CI: 1.109-13.56).CD8+T cells increased after therapy and CTC decreased also showed longer PFS(p<0.05)..3.To further explore the mechanism of low dose radiotherapy on T cell recruitment,we first observed that irradiated tumor cells promote T cells migrating from the Transwell upper chamber to the lower chamber.Single tumor mouse model showed an increase in T cell recruitment after low dose radiotherapy.RNA transcriptome sequencing,fluorescence quantitative PCR and flow cytometry verification showed that the expression of chemokine CXCL9,CXCL10,CXCL11 and receptor CXCR3 was upregulated after low dose radiotherapy(p<0.05).In order to observing whether the inhibition of chemokine receptor inhibits the recruitment of T cells induced by low-dose radiotherapy low dose radiation combined with CXCR3inhibitor(αCXCR3)were administrated.Flow cytometry and immunofluorescence detection results showed that αCXCR3 inhibited the recruitment of CD8+T cells in tumor induced by low dose radiotherapy(p<0.05).Western blot detection showed that the expression levels of IFN-γ and TNF-α protein in tumor after αCXCR3combined with low dose radiotherapy were lower than those in the low-dose radiotherapy alone group.αCXCR3 also weakened the control of low-dose radiotherapy on tumor growth in mice(p < 0.05).Finally,low dose radiotherapy combined with high-dose radiotherapy and PD-1 inhibitor in the dual-tumor mouse model also showed that the recruitment of T cells and the expression of chemokines CXCL9,CXCL10 and CXCL11 in distant tumors increased after low dose radiotherapy(p<0.05).Conclusion:The triple therapy mode of low dose radiotherapy combined with SBRT and PD-1inhibitors was safe as first line in the treatment of PD-L1 positive advanced NSCLC patient,and the triple therapy showed positive response.Triple therapy may activate circulating immune T cells and reduce circulating tumor burden.Mouse models suggest that low dose radiotherapy may induce T cell recruitment into the tumor by up-regulating the expression of chemokine CXCL9,10,11 and its receptor CXCR3.